The development of the anogenital system requires precise regulation of growth and patterning, and defect in these processes result in several congenital birth defects. These range from hypospadias, a failure in urethral tube closure resulting in ectopic urethral openings, to persistent cloaca, in which a single outlet exists for the alimentary and urogenital systems. Despite the incidence of anorectal malformations associated with problems during development, very little is known about the embryology and molecular regulation of the development of the anogenital system. The goal of this project is to define the role of FoxAl and FoxA2, two transcription factors involved in endoderm development and differentiation, in the development of the anogenital system. Our specific aims are to (1) Test the hypothesis that early FoxA1 and FoxA2 are required for development of the anogenital system. We will determine the morphological and developmental causes for persistent cloaca in FoxAl;FoxA2 conditional compound mutant mice. In addition, we intend to determine whether cell fate in cloaca is maintained in the absence of FoxAl and FoxA2. (2)Test the hypothesis that late FoxAl and FoxA2 are required for patterning the external genitalia. We will delete FoxA2 in the developing genitalia during later patterning stages to determine temporal role of FoxAl and FoxA2 in morphogenesis of the urethra. To test the hypothesis that FoxAl and FoxA2 initiate molecular patterning within the external genitalia, we will examine FoxAl;FoxA2 conditional mutants for changes in expression of patterning genes expressed in the genital tubercle. Finally, we will determine the genetic relationship between FoxAl and FoxA2, and Sonic Hedgehog (Shh). Shh is critical in development of the external genitalia, and a genetic relationship has been established between Shh and FoxAl and FoxA2 in other endodermally-derived tissues. We will test the hypothesis that FoxAl and FoxA2 regulate expression and function of Shh in the tubercle by examining the expression of Shh in FoxAl;FoxA2 conditional mutants. The experiments proposed in this research proposal have important implications for understanding the basis of human anogenital birth defects.